Recombinant L-methionine γ-lyase

ABSTRACT

A recombinant protein capable of constituting a recombinant L-methionine γ-lyase or a variant thereof, a recombinant oligomeric enzyme, preferably tetrameric enzyme, consisting of the recombinant protein, a DNA molecule encoding the same, an expression vector containing the DNA molecule, a host cell harboring the expression vector, a method of preparing the recombinant L-methionine γ-lyase, and anti-tumor agent comprising the recombinant L-methionine γ-lyase or a variant thereof are provided.

FIELD OF THE INVENTION

The present invention relates to a recombinant L-methionine γ-lyase.More particularly, it relates to a recombinant protein capable ofconstituting a recombinant L-methionine γ-lyase or a variant thereof, arecombinant oligomeric enzyme consisting of the recombinant protein orits variant, a DNA molecule encoding the same, an expression vectorcontaining the DNA molecule, a host cell harboring the expressionvector, a method of preparing the recombinant L-methionine γ-lyase ofthe present invention by culturing the host cell, and anti-tumor agentcomprising the recombinant L-methionine γ-lyase or a variant thereof.

BACKGROUND OF THE INVENTION

L-methionine γ-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxalphosphate as a co-enzyme and catalyzes α, γ-elimination andγ-replacement of L-methionine or its derivatives and also α,β-elimination and β-replacement of S-substituted L-cysteine or itsderivatives Tanaka, H. et al., Biochemistry, 16, 100-106 (1977)!. Thisenzyme has been isolated and purified mainly from Pseudomonas putida andits physicochemical and enzymological properties have already been shownNakayama, T. et al., Anal. Biochem., 138, 421-424 (1984)!. There havebeen some researches reporting the mechanism of enzymatic reactioncatalyzed by L-methionine γ-lyase Esaki, N. et al., FEBS Lett., 84,309-312 (1977); Nakayama, T. et al., Biochemistry, 27, 1587-1591 (1988),and the like!. The references which have been provided so far are allrelated to L-methionine γ-lyases purified from natural source such asbacteria belonging to genus Pseudomonas. Thus, there are no referencesdescribing or suggesting the preparation of a recombinant enzyme bymeans of genetic engineering.

Recently, it has been suggested that naturally occurring L-methionineγ-lyase purified from a culture of P. putida may have an anti-tumoractivity WO94/11535, Publication date, May 26, 1994).

To develop a therapeutically applicable and useful drug, a substance tobe used as an active ingredient should be pure or essentially free fromundesirable contaminants, for example, proteins or the like by which thesubstance is naturally accompanied, and can be produced on a large scaleso as to meet the demands. Accordingly, the establishment of a method ofpreparing L-methionine γ-lyase or its variants with essentially the sameenzymatic activity by means of recombinant DNA technique must contributeto the development of useful anti-tumor agents.

WO95/17908 has revealed production of L-methionine γ-lyase using generecombinant technique, and describes the DNA sequence and the deducedamino acid sequence L-methionine γ-lyase. However, the document showsneither any primers for isolating the DNA encoding L-methionine γ-lyasenor any source from which the DNA comes.

SUMMARY OF THE INVENTION

The present inventors have succeeded in isolating and identifying DNAencoding L-methionine γ-lyase from a strain of Pseudomonas putida,cloning and expression of the same in Escherichia coli and preparing therecombinant enzyme using the same.

The enzyme L-methionine γ-lyase is known to be an oligomeric enzyme. Aswill be described in detail below, four units of the recombinant proteinof the present invention, when it is produced in a recombinant straintransformed with an expression vector containing a DNA encoding the saidprotein, combine spontaneously to form the oligomeric enzyme withL-methionine γ-lyase activity. Thus, the protein of the presentinvention having amino acid sequence shown in SEQ ID NO:1, although itdoes not have any biological activity by itself, is a subunit ofL-methionine γ-lyase which is expected to be useful as anti-tumor agentas mentioned above.

Accordingly, in the present specification, the terms "recombinantL-methionine γ-lyase" and "recombinant protein" encoded by DNA havingnucleotide sequence shown in SEQ ID NO: 1 and a derivative thereofcapable of constituting oligomeric enzyme having L-methionine γ-lyaseactivity may be used exchangeably.

DETAILED DESCRIPTION OF THE INVENTION

One of strains of P. putida capable of providing a DNA encoding aprotein constituting a recombinant L-methionine γ-lyase of the presentinvention, P. putida ICR 346 has been deposited at the "NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology", at Higashi 1-1-3, Tsukuba-shi, Ibaraki-ken,Japan under the Budapest Treaty (accession number: FERM BP-5238;deposition date: Sep. 25, 1995). The mycological characteristics ofPseudomonas putida ICR 3460 are shown below.

1. Morphology

It is a Gram-negative aerobic rods motile by one or several polarflagella, and dose not grow under anaerobic conditions. Its size ranges0.7 to 1.0 μm in diameter and 2.0 to 3.5 μm in length.

2. Characteristics of Culture

(1) Nutrient agar plate culture (at 30° C. for 24 to 168 hours)

Growth rate: Colonies were formed after 24 hours.

Form: Circular

Surface: Smooth

Margin: Entire

Elevation: Raised

Chromogenesis: Pale yellow

Lustre: Glistening

Optical characters: Opaque

(2) Nutrient agar slant culture (at 30° C. for 24 to 168 hours)

Growth: Abundant

Form of growth: Spreading

Surface: Smooth

Chromogenesis: Pale yellow

Lustre: Glistening

Optical characters: Opaque

Consistency: Butyrous

(3) Nutrient broth liquid culture (at 30° C. for 24 to 168 hours)

Surface growth: Membranous

Clouding: Moderate

Odor: Decided

Sediment: Compact

Amount of sediment: Abundant

3. Biochemical Properties

(1) Nitrate reduction: Negative

(2) Methyl red test: Negative

(3) Voges-Proskauer test: Negative

(4) Indole production: Negative

(5) H₂ S production: Negative

(6) Starch hydrolysis: Negative

(7) Citrate utilization: Positive

(8) Production of pigment: Water-soluble fluorescent yellow-greenpigment was produced.

(9) Urease: Negative

(10) Oxidase: Positive

(11) Catalase: Positive

(12) OF-test: Sugars were decomposed by oxidation.

(13) Utilization of carbon compounds

i) D-Glucose: Positive

ii) L-Arabinose: Negative

iii) D-Mannose: Negative

iv) D-Mannitol: Negative

v) Maltose: Negative

vi) N-Acetylglucosamine: Negative

vii) Potassium gluconate: Positive

viii) N-Capric acid: Positive

ix) Adipic acid: Negative

x) DL-Malic acid: Positive

xi) Phenylacetate: Positive

(14) Arginine dihydrolase: Positive

(15) Esculin hydrolysis: Negative

(16) Gelatin liquefaction (hydrolysis): Negative

(17) Deoxyribonuclease (DNase): Negative

(18) β-Galactosidase (PNPG test): Negative

(19) Acylamidase: Negative

(20) Growth under anaerobic conditions: Negative

(21) Behavior for oxygen: Aerobic

The bacterial strain was identified as a strain of Pseudomonas putida bycomparing its taxonomic properties with the taxonomic description aboutP. putida in Bergey's Manual of Systematic Bacteriology Vol. 1 (1984),and designated as Pseudomonas putida ICR 3460.

The present invention also provides a method of preparing recombinantL-methionine γ-lyase, which comprises constructing a recombinant vectorby introducing a DNA sequence encoding the amino acid sequence shown inSEQ ID No. 1 into a vector used in the host/vector system for expressionin E. coli, transforming an E. coli host cell with the vector, andculturing the transformant. The present method can be carried outaccording to the steps as exemplified below. As is easily understood byone ordinary skilled in the art, once the DNA sequence codingL-methionine γ-lyase is disclosed by the present invention, certainsteps can be omitted or simplified.

(1) A strain of P. putida is grown overnight at 28° C. in a suitablemedium. Example of medium includes those obtainable by addingL-methionine to a conventional medium in which a species of Pseudomonascan generally grow. From the resulting culture broth, cells areharvested and mechanically disrupted. After the cell debris wasdiscarded by centrifugation, the supernatant is subjected to dialysisfollowed by ion exchange chromatography (2 times) and the like to purifyL-methionine γ-lyase. The purified L-methionine γ-lyase is digested witha suitable chemical reagent or enzyme generally used for fragmentationof protein. Peptide fragments are separated into respective ones and oneof them is subjected to the amino acid sequencing from the aminoterminus.

(2) A base sequence of a DNA capable of encoding a portion of the aminoacid sequence of the peptide fragment of the purified L-methionineγ-lyase protein is deduced. Oligonucleotides corresponding to thededuced base sequences are chemically synthesized, labeled at its5'-terminus with ³² P and used as a probe for gene cloning.

(3) The chromosomal DNA is extracted from P. putida, digested with anappropriate restriction endonuclease and subjected to electrophoresis onagarose gel. The relevant region on the agarose gel containing the DNAfragment encoding L-methionine γ-lyase protein is excided and the DNA isextracted.

(4) The DNA obtained in the step (3) is inserted into a cloning vectorfor E. coli and the resultant recombinant vector is intorduced into astrain of E. coli, for example E. coli strain MV1184, JM109 or the like.The transformant is cultured so as to form colonies or plaques on anagar medium. Then, colony or plaque hybridization is carried out usingthe ³² P labeled probe, and the colony or plaque showing homology withthe probe is selected and isolated.

(5) The recombinant plasmid is extracted from the selected E. colistrain or phage particles. After the restriction enzyme map is prepared,the base sequence of the cloned DNA fragment derived from P. putida isdetermined. The amino acid sequence deduced from thus determined DNAbase sequence is compared with known partial amino acid sequences,terminal amino acid sequence and amino acid composition of L-methionineγ-lyase. Finally, the base sequence of the structural gene ofL-methionine γ-lyase is determined.

(6) An expression plasmid is constructed by inserting the structuralgene of L-methionine γ-lyase into an expression vector for E. coli sothat the gene is located downstream from an E. coli promoter.

(7) The expression plasmid is then introduced into E. coli host cells toobtain novel E. coli transformants capable of producing L-methionineγ-lyase.

One ordinary skilled in the art can easily carry out general operationsrequired for handling DNA in the above steps in accordance with theexperimental mannals, for example, "Molecular Cloning" 2nd editionSambrook, J. et al., Cold Spring Harbor Laboratory (1989)!. All theenzymes, reagents and the like used in the above steps are commerciallyavailable. Unless otherwise noted, one can achieve the purposecompletely just by using them under the conditions as instructed by themanufacturers.

In the step (1), the method of amino acid sequencing is known and can becarried out by means of, for example, a commercially available automaticamino acid sequencer. In the step (2), oligonucleotides having anintended base sequence can be synthesized by the use of a commerciallyavailable DNA synthesizer according to the indicated operationprocedures. In the step (5), the DNA sequencing can be carried outaccording to the known method of Sanger et al., Proc. Natl. Acad. Sci.U.S.A., 74, 5463-5467 (1977), using M13 vector system.

Example of E. coli vectors used for cloning in the step (4) includesplasmid vectors such as pUC118, pUC119, pUC13, pBR322, pAT153 and thelike and phage vectors such as λZAPII, λgt10 and the like.

Example of E. coli strains usable as a host for cloning and expressionincludes strains of E. coli K-12 such as HB101, DH1, C600, JM103, JM105,JM109, MV1184 and the like. The above-mentioned vectors and hosts aremarketed and available in ease.

When yeast is used as a host for expression, the objective recombinantL-methionine γ-lyase can be separated from the supernatant of culturebroth easily. In this case, vectors such as pYES2, pYEUra3 and the likecan be used. It is possible to carry out so-called self-cloning by theuse of a strain of genus Pseudomonas bacteria as a host. In this case,vectors such as RSF1010, RK2 and the like are used.

In the step (6), a plasmid capable of directing an efficient expressionof a gene of interest in E. coli can be constructed by inserting a DNAfragment containing the intended structural gene of L-methionine γ-lyaseinto an expression vector such as pKK223-3, pPL-lambda or the like whichcomprise suitable promoters (e.g., Lac, Tac, Trp, λP_(L) or the like)functional in the host together with Shine-Dalgarno (SD) sequence, orATG vector such as pKK233-2, pTrc99A or the like which further comprisesthe translational initiation codon ATG. An efficient expression can beachieved by introducing the resultant expression plasmid into a suitablehost such as E. coli strain JM103, JM109, HB101, C600 or the like.

The expression product, L-methionine γ-lyase, can be purified usingconventional methods including centrifugation, gel filtration, ionexchange chromatography and the like, alone or in an appropriatecombination.

The recombinant enzyme obtained by the present method has been proved tohave L-methionine γ-lyase activity. It has also been confirmed that theactivity is substantially unchanged even if methionine at the N-terminusof the amino acid sequence of the resultant recombinant L-methionineγ-lyase is absent.

As used herein, the term "variants of recombinant proteins" refers toproteins feasible to either chemical or biochemical modification, orthose having naturally or artificially modified amino acid or amino acidsequence, on condition that they have substantially the same functionsor activity as those of the recombinant protein of the present inventionhaving amino acid sequence shown in SEQ ID NO: 1. Such variants can beprepared by methods well known in the art, for example, site-directedmutagenesis by PCR method and the like.

The variants are exemplified by proteins in which the amino acidsequence is slightly different from No. 2 to No. 398 of that shown inSEQ ID NO: 1, induced by minor alteration such as addition, deletionand/or substitution of an amino acid or an amino acid sequence and whichhave substantially the same functions or activities as those of therecombinant protein of the present invention comprising the amino acidsequence from No. 2 to No. 398 of the shown in SEQ ID NO:1. Preferably,such variants include the proteins shich contain one or morealteration(s) as shown above of one or a few amino acid(s). Morepreferably, the variants include proteins having 85% or more homology inthe amino acid sequence to the recombinant protein of the presentinvention.

The present invention also provides DNA molecules encoding suchvariants. Preferably, the DNA molecules include the nucleotide sequenceswhich encode the variants and which are analogous to that of No. 64 toNo. 1254 of that shown in SEQ ID NO;1.

As will be understood from the specification, when a strain of E. colitransformed with an expression vector of the present invention is grownin an appropriate medium, the expressed recombinant protein insolubilized fraction (cell-free extract) by itself forms the oligomer,thereby providing a biologically active recombinant enzyme. Theresultant oligomer, preferably tetramer of the recombinant protein ofthe present invention or a variant thereof is useful as an anti-tumoragents see, WO94/11535!.

Accordingly, the present invention further provides an anti-tumor agentcomprising the recombinant L-methionine γ-lyase of the present inventionor variants thereof. The anti-tumor agent of the present invention canbe prepared using any of known carriers or excipients therefor. It istypically in the form of injections and can be lyophilized products. Foradministration, it will be generally infused parenterally every day overseveral ten minutes to several hours for several days to several weeks.Although the administration dose varies depending on intended effects,sex, age, weight and severity of symptoms of patients and the like, itis generally from about 1 to 1000 U/kg weight/day, and preferably fromabout 50 to 500 U/kg weight/day.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows the N-terminal amino acid sequence of native L-methionineγ-lyase purified from P. putida, wherein the partial sequence used forpreparation of oligonucleotide probes used in the isolation of DNAencoding L-methionine γ-lyase is underlined. It is also shown nucleotidesequences which can encode the partial amino acid sequence and is usableas oligonucleotide probes.

FIG. 2 is a flow chart illustrating the procedures for constructing theexpression plasmid pYH103.

FIG. 3 is a flow chart illustrating the procedures for constructing theexpression plasmid pYH301.

The following Examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Unless otherwise stated, throughout the Examples below, the digestionreaction using a restriction endonuclease was carried out at 37° C. for2 hours in an indicated buffer according to the instructions of themanufacturers.

Further, the ligation reaction using T4 DNA ligase was carried out at16° C. for 16 hours in an indicated buffer according to the instructionsof the manufacturer.

Transformation of E. coli with a plasmid was carried out according tothe method of Cohen et al. (Cohen et al., Proc. Natl. Acad. Sci. USA,69, 2110-2114 (1972)).

Example 1 Synthesis of DNA Probes and End-Labeling

Pseudomonas putida was grown at 28° C. in an L-methionine-containingproduction medium (0.25% L-methionine, 0.1% polypeptone, 0.1% glycerol,0.1% KH₂ PO₄, 0.1% K₂ HPO₄, 0.01% MgSO₄ ·7H₂ O, 0.025% yeast extract, pH7.2) according to the procedures described by Nakayama, T. et al, Anal.Biochem, 138., 421-424 (1984). Native L-methionine γ-lyase was purifiedfrom cells of P. putida according to the known procedures Nakayama, T.et al, Anal. Biochem., 138, 421-424 (1984); and Nakayama, T. et al.,Biochemistry, 27, 1587-1591 (1988)!. The purified enzyme wasfragmentated with cyanogen bromide and a fragment containing theN-terminal amino acid sequence was isolated and subjected to thedetermination of amino acid sequence by Edman's procedures using anautomated amino acid analyzer. The amino acid sequence from residue 15isoleucine to residue 21 proline from the N-terminus was as follows:

    Ile His His Gly Tyr Asp Pro (SEQ ID NO: 3)

The sequence above has not been described in literatures.

The N-terminal amino acid sequence is shown in FIG. 1 together withnucleotide sequences which can encode a partial amino acid sequenceindicated by underline, and be used a probe.

Thus, various oligonucleotides which can encode the amino acid sequenceabove were synthesized with a DNA synthesizer Model 391 (AppliedBiosystems) as shown in FIG. 1. The DNA probe thus obtained was labeledat its 5' terminus using T4 polynucleotide kinase and γ-³² P!ATP (220TBq/mmol) New Research Products! according to the known method Maxam, A.M. and Gilbert, W., Methods Enzymol., 65, 499 (1980)!.

Example 2 Cloning of DNA Fragment Encoding L-Methionine γ-Lyase Protein

1. Extraction of Chromosomal DNA from P. putida

P. putida ICR3460 (FERM BP-5238) was grown in LB medium overnight andcells were harvested from the culture (4 g, wet weight). After washing(2 times) with Nacl-EDTA (0.15M NaCl, 0.1M EDTA (pH 8.0)), the cellswere suspended into Nacl-Tris (0.1M NaCl, 0.1M Tris-HCl (pH 9.0), 0.01MEDTA (pH 7.5)), and a portion (9 ml) of the suspension was placed in arotor tube (30 ml capacity) to give about 1 g cells/tube. To thesuspension was added 1 ml of 20 mg/ml lysozyme (Nacalai Tesgue) solutionin Nacl-EDTA and the mixture was incubated at 37° C. for 20 minutes.After addition of 3 ml Tris-SDS 5% SDS in Nacl-Tris-EDTA (0.14M NaCl, 20mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0))!, the tube was gently invertedto suspend the mixture and then maintained at 60° C. for 20 minutes.After allowing the tube to cool to room temperature, 2.5 ml of 1.5 mg/mlproteinase K (Wako-Pure Chemical) was added and incubated for another 10hours. To the mixture was added an equal volume of phenol solution(phenol saturated with 100 mM Tris-HCl (pH 8.0)) and the tube wasallowed to stand for 30 minutes with occasional and gentle shaking,followed by centrifugation at 3000 rpm for 10 minutes. The contents inaqueous phase were combined in a flask containing cold ethanol. Theprecipitated DNA was wound around a glass stick, washed successivelywith 70%, 80%, and 90% ethanol, suspended in 20 ml 0.1x SSC (0.01M NaCl,0.0015M sodium citrate). After the phenol treatment was repeated, 100 μlof 10 mg/ml RNaseA was added to the DNA solution and the mixture wasincubated at 37° C. for 1 hour. After repeating the phenol treatmentonce more, the DNA was suspended in 15 ml NaCl-Tris-EDTA and centrifugedat 10,000 rpm for 10 minutes. The supernatant was dialyzed against TEbuffer (10 mM Tris-HCl (pH 7.5)-1 mM EDTA (pH 8.0)) overnight. (Thedialyzing tube had been treated by boiling in a mixture of 1 mM EDTA (pH8.0) and 2% Na₂ CO₃ for 10 minutes, washing with water and boiling againin 1 mM EDTA (pH 8.0) for 10 minutes). To the dialyzate was added 25 μlchloroform to obtain a DNA preparation. The purity of DNA was determinedby calculating the ratio of absorbance at 260 nm to that at 280 nm. As aresult, from 4 g (wet weight) cells was yielded 5 mg DNA in total.

To 80 μg of the DNA was added 560 units of restriction endonuclease PstIand reaction was carried out under an optimal condition. The resultantreaction mixture was electrophoresed on agarose gel in a conventionalmanner and subjected to Southern blot hybridization according toSouthern's method Southern, E. M., Methods Enzymol., 68, 152-176 (1979)!using the oligonucleotide probe end-labeled with ³² P prepared inExample 1. The region of the gel corresponds to around 1 kb where thespecific signal was detected was cut. The DNA fragments were extractedfrom the gel according to the method of Vogelstein and Gillespie, Proc.Natl. Acad. Sci. USA, 76, 615-619 (1979).

2. Cloning of PstI Fragment

Plasmid pUC118 (1.5 μg, Takara Shuzo) was digested with 120 units ofrestriction endonuclease PstI. To the reaction solution (2 μl)containing digested pUC118 were added 2 μg of a mixture containing PstIrestriction fragments obtained in step 1 above and 1000 units of T4 DNAligase (Nippon Gene) to ligate the PstI fragments into plasmid pUC118.The resultant mixture containing recombinant plasmids were used totransform E. coli MV 1184 (Takara Shuzo), and the transformants werespread over L-agar plate (1% polypeptone, 0.5% yeast extract, 1% NaCl,1.5% agar) containing 50 μg/ml ampicillin and incubated overnight at 37°C. Colonies of transformants were transferred to nylon membrane (tradename; Hybond N+, Amersham) and the DNA derived from colonies wereimmobilized on the membrane according to a method well-known in the art.

Transformants were screened by colony hybridization using ³²P-end-labeled oligonucleotide probes prepared in Example 1 (Hanahan etal., Gene, 10, 63-67, (1980)). As a result, one positive clone harboringthe recombinant plasmid pMR1 was obtained.

The DNA base sequence analysis by the dideoxy chain termination method(Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977))revealed that the plasmid pMR1 contains a 931 bp of PstI insert whichcontains only 422 bp fragment of the 5' terminal region of L-methionineγ-lyase gene.

3. Construction of Second Genomic Library

Prior to the re-cloning, an oligonucleotide probe corresponding toHindIII-PstI region containing 422 bp of 5' terminal region ofL-methionine γ-lyase gene was prepared using plasmid pMR1 obtained instep 2 above as follows.

A 453 bp HindIII-PstI fragment was obtained from plasmid pMRI (30 μg)through the digestion with 300 units of restriction endonucleaseHindIII, ethanol precipitation to terminate the reaction, and digestionwith 300 units of restriction endonuclease PstI. The resultant DNAfragment was labeled with α-³² P!dCTP (220 TBq/mmol; New ResearchProducts) using random primer labeling kit (Takara Shuzo).

The chromosomal DNA was then extracted from P. putida ICR3460 (FERMBP-5238) in a manner similar to that described in step 1 above. The DNA(40 μg) was digested with 75 units of restriction endonuclease SacI andthe resultant reaction mixture was electrophoresed on agarose gel in aconventional manner. Then, Southern blot hybridization was carried outusing the end-labeled oligonucleotide probes obtained in step 3 above.The DNA fragment around 2.6 kb where the specific signal was detectedwas extracted from the gel.

Cloning phage vector λZAPII (2.3 μg; Stratagene) was digested with 20units of SacI in a similar manner. The both of the SacI fragments wereligated with T4 DNA ligase and packaged in the following manner usingLAMBDA INN in vitro packaging kit (Nippon Gene). To a tube containingpackaging extract was added 1 μl (0.1 μg) λZAPII phage DNA and themixture was incubated at 22° C. for 2 hours. After addition of 500 μl ofSM buffer (0.58% NaCl, 0.2% MgSO₄ ·7H₂ O, 50 mM Tris-HCl (pH 7.5), 0.01%gelatin), the mixture was gently mixed and a drop of chloroform wasadded to obtain a phage solution. E. coli strain XL1-Blue was inoculatedinto 5 ml LB medium containing 0.2% maltose and 10 mM MgSO₄ andincubated overnight. One milliliter each of the culture broth wascharged in an Eppendorf tube and centrifuged at 3,000 rpm for 7 minutes.The supernatant was discarded and the cell pellet was suspended into200-300 μl of 10 mM MgSO₄. An aliquot (100 μl) of the cell suspensionwas charged in a tube for phage infection. The cell suspension wasincubated with 10 μl of phage solution diluted with SM buffer at 37° C.for 20 minutes. The infected bacteria were inoculated into a mixtureprepared by adding 50 μl each of 0.5M isopropyl-β-thiogalactopyranoside(IPTG) and 125 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal)to 3 ml of NZY upper-layer agarose medium (1% NZ amine (casein enzymatichydrolysate), 0.5% NaCl, 0.5% yeast extract, 0.2% MgSO₄ ·7H₂ O, 0.7%agarose, pH 7.5) which was previously warmed and maintained at 48° C.,and then the mixture was overlaid on NZY agarose medium (1% NZ amine(casein enzymatic hydrolysate), 0.5% NaCl, 0.5% yeast extract, 0.2%MgSO₄ ·7H₂ O, 1.5% agarose, pH 7.5). Incubation was carried out at 37°C. for 6-7 hours after the medium surface dried to form plaques.

Plaque hybridization was carried out according to the method of Benton,W. D. and Davis, A. W., Science, 196, 180-182 (1977). Plaques formed ona plate were transferred onto Hybond N+ nylon membrane, and DNA derivedfrom plaque was immobilized on the membrane with 0.4N NaOH, hybridizedwith previously prepared probes, and followd by autoradiography. As aresult, two positive clones were obtained. The recombinant phage DNAseach containing cloned DNA fragment were purified according to themethod of Yamamoto et al., Virology, 40, 734 (1970). Restrictionendnuclease analysis of the DNA inserts in the positive clones revealedthat the both clones have the same 2.6 kbp SacI fragment.

4. Construction of Cloning Vector

Cloning vector pUC118 (3 μg) was digested with 20 units of restrictionendonuclease SacI and ligated to 2.6 kbp SacI fragment obtained in step3 above with 1000 units of T4 DNA ligase to yield plasmids named pYH1and pYH2.

The plasmids pYH1 and pYH2 were used to transform E. coli strain MV1184.The transformants were grown and analyzed for the expression ofL-methionine γ-lyase activity.

E. coli strain MV1184 transformed with either plasmid was incubated inL-broth containing 50 μg/ml ampicillin and 1 mM IPTG at 37° C. for 17hours. From cells was prepared 2.5 ml of cell-free extract according tothe method of Nakayama, T. et al, Anal. Biochem., 138, 421-424 (1984)and subjected to an enzyme assay. The assay was carried out using anassay system containing 200 μmol of potassium phosphate buffer (pH 8.0),40 μmol of L-methionine, 0.04 μmol of pyridoxal 5'-phosphorate andcell-free extract in a final volume of 1.6 ml. After incubating themixture at 37° C. for 5 minutes, 0.2 ml of 50% trichloroacetic acid wasadded to terminate the reaction. The reaction mixture was centrifugedand the reaction product α-ketobutyrate in the supernatant wasdetermined using 3-methyl-2-benzothiazolinone hydrazone according to themethod of Soda, K., Anal. Biochem., 25, 228-235 (1968). The protein wasdetermined according to the method of Lowry et al., J. Biol. Chem., 193,265-275 (1951) using bovine serum albumin as a standard.

The assay revealed that the cell extract from E. coli transformantsharboring plasmid pYH1 has L-methionine γ-lyase activity while thatobtained from transformants harboring plasmid pYH2 does not have theactivity at all as shown in Table 1 below. The results showed that theplasmids pYH1 and pYH2 differ from each other in the orientation ofinserted SacI fragment. That is, the insert is in the same or reverseorientation with respect to lac promoter in pUC118.

Example 3 DNA Sequence Analysis

The plasmid pYH1 prepared in Example 2 was used for DNA sequenceanalysis to determine the base sequence of a DNA region encodingL-methionine γ-lyase and its flanking regions.

DNA fragments corresponding to each region to be sequenced were obtainedfrom plasmid pYH1 and subcloned into vector pUC118 or pUC119. By meansof infection of helper phage M13KO7, each single-stranded DNA wasprepared according to the method of Viera et al., Methods Enzymol., 153,3-11 (1987). The sequencing was carried out using the single-strandedDNA as a template, M13 sequence primer labeled with γ-³² P!ATP (220TBq/mmol: NEN Research Products) and BcaBEST sequencing kit (TakaraShuzo) by the dideoxy chain termination method. The determined basesequence is provided in SEQ ID NO: 1, which contains one open readingframe of 1194 nuclotides in total length starting with ATG at positions61-63. This region encodes a protein of 398 amino acid residues and wasidentified as structural gene of L-methionine γ-lyase on the basis ofthe facts below.

a) The N-terminal amino acid sequence is in agreement with that ofpreviously determined sequence of L-methionine γ-lyase (Nakayama, T. etal., Biochemistry, 27, 1587-1591 (1988));

b) the presumed amino acid composition of the protein enencoded is ingood agreement with that of L-methionine γ-lyase (Nakayama, T. et al.,ibid.); and

c) the calculated molecular weight (about 43 kDa) is in agreement withthat of L-methionine γ-lyase subunit (Nakayama, T. et al., Anal.Biochem., 138, 421-424 (1984)).

Example 4 Construction of Expression Vector

Recombinant E. coli MV1184/pYH1, when grown in L-broth while adding IPTGat a final concentration of 1 mM, expressed L-methionine γ-lyaseefficiently. An expression vector capable of directing higher expressionin E. coli was constructed as follows.

A 1.5 kb SalI fragment was isolated by partially digesting 20 μg of pYH2with 10 U of SalI. The 1.5 kb SalI fragment was then ligated to a 3.14kb fragment obtained by digesting 3 μg of pUC119 (Takara Shuzo) with 20U of SalI in the presence of 1000 U of T4 DNA ligase to yield pYH101.The pYH101 (2 μg) was digested with 200 U of HindIII to obtain a 4.5 kbfragment, which was then self-ligated with 1000 U of T4 DNA ligase andtransformed into E. coli strain MV1184. Transformants were selected byincubating on L-agar containing 50 μg/ml ampicillin overnight at 37° C.A recombinant plasmid named pYH103 was isolated from the intendedtransformant. FIG. 2 shows the restriction enzyme cleavage map ofplasmid pYH103.

The plasmid pYH103 was subjected to the assay for the ability to directthe expression of L-methionine γ-lyase activity in a manner similar tothat described in Example 2, step 4 above. The results are summarized inTable 1 below.

                  TABLE 1    ______________________________________               Specific Activity (U/mg protein)               LB     LB (IPTG)    ______________________________________    pYH1         0.008    0.39    PYH2         NA       --    pYH103       --       0.71    ______________________________________     Note:     One unit of the enzyme is defined as the amount of enzyme that catalyzes     the formation of 1 μmol of ketobutyrate at 37° C. per minute.     LB means Lbroth and LB (IPTG) means Lbroth containing 1 mM IPTG.     "NA" and "--" mean "no activity" and "not assayed", respectively.

Example 5 Construction of Plasmid with High Expression Efficiency

Plasmid pYH103 (5 μg) prepared in Example 4 was digested with 30 unitsof HindIII restriction endonuclease and precipitated with ethanol, whichwas followed by digestion with 30 units of BamHI. After agarose gelelectrophoresis, a 1.3 kbp HindIII-BamHI fragment containing thestructural gene of L-methionine γ-lyase was extracted from the gel andpurified, and dissolved in 20 μl TE buffer (10 mM Tris-HCl (pH 7.5), 1mM EDTA (pH 8.0)). The resultant fragment was then blunt-ended by theaddition of 2 units of Klenow enzyme (Nippon Gene).

Plasmid pKK223-3 (3 μg, Pharmacia Biotech) was digested with 30 units ofrestriction endonuclease SmaI. To the reaction solution (3 μl), thesolution (10 μl) containing the blunt-ended fragment above and 1000units of T4 DNA ligase were added and incubated for ligation to obtainplasmid pYH301. The restriction enzyme cleavage map of the resultantplasmid pYH301 is provided in FIG. 3.

The plasmid pYH301 was assayed to evaluate the expression efficiencyaccording to the method described in Example 2, step 4 except that E.coli strain JM109 (Takara Shuzo) was used as a host cell. The assayrevealed that a cell free extract obtained from the transformantharboring plasmid pYH301 grown in L-broth containing 1 mM IPTG at 37° C.for 17 hours shows a specific activity of 0.82 U/mg.

What is claimed is:
 1. A recombinant protein comprising an amino acidsequence of residue from No. 2 to residue No. 398 of SEQ ID NO:1,wherein said protein is free of other proteins from P. putida or avariant thereof.
 2. The recombinant protein as claimed in claim 1,wherein the amino acid sequence further contains a methionine residueattached at its N-terminus.
 3. The recombinant protein as claimed inclaim 1 or 2, wherein the amino acid sequence is encoded by DNAoriginated from a strain of Pseudomonas putida.
 4. The recombinantprotein as claimed in claim 1 or 2, wherein the amino acid sequence isencoded by DNA originated from Pseudomonas putida ICR3460 (FERMBP-5238).
 5. An oligomeric enzyme comprising as subunit the recombinantproteins as claimed in claim 1 or
 2. 6. The enzyme as claimed in claim5, which is a tetramer.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1320 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 61..1254    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TGGAAAAATTTAAGCCGGTCTGTGGAATAAGCTTATAACAAACCACAAGAGGCGGTTGCC60    ATGCACGGCTCCAACAAGCTCCCAGGATTTGCCACCCGCGCCATTCAC108    MetHisGlySerAsnLysLeuProGlyPheAlaThrArgAlaIleHis    151015    CATGGCTACGACCCCCAGGACCACGGCGGCGCACTGGTGCCACCGGTC156    HisGlyTyrAspProGlnAspHisGlyGlyAlaLeuValProProVal    202530    TACCAGACCGCGACGTTCACCTTCCCCACCGTGGAATACGGCGCTGCG204    TyrGlnThrAlaThrPheThrPheProThrValGluTyrGlyAlaAla    354045    TGCTTTGCCGGCGAGCAGGCCGGGCATTTCTACAGCCGCATCTCCAAC252    CysPheAlaGlyGluGlnAlaGlyHisPheTyrSerArgIleSerAsn    505560    CCCACCCTCAACCTGCTGGAAGCACGCATGGCCTCGCTGGAAGGCGGC300    ProThrLeuAsnLeuLeuGluAlaArgMetAlaSerLeuGluGlyGly    65707580    GAGGCCGGGCTGGCGCTGGCCTCGGGCATGGGGGCGATCACGTCCACG348    GluAlaGlyLeuAlaLeuAlaSerGlyMetGlyAlaIleThrSerThr    859095    CTATGGACACTGCTGCGCCCCGGTGACGAGGTGCTGCTGGGCAACACC396    LeuTrpThrLeuLeuArgProGlyAspGluValLeuLeuGlyAsnThr    100105110    CTGTACGGCTGCACCTTTGCCTTCCTGCACCACGGCATCGGCGAGTTC444    LeuTyrGlyCysThrPheAlaPheLeuHisHisGlyIleGlyGluPhe    115120125    GGGGTCAAGCTGCGCCATGTGGACATGGCCGACCTGCAGGCACTGGAG492    GlyValLysLeuArgHisValAspMetAlaAspLeuGlnAlaLeuGlu    130135140    GCGGCCATGACGCCGGCCACCCGGGTGATCTATTTCGAGTCGCCGGCC540    AlaAlaMetThrProAlaThrArgValIleTyrPheGluSerProAla    145150155160    AACCCCAACATGCACATGGCCGATATCGCCGGCGTGGCGAAGATTGCA588    AsnProAsnMetHisMetAlaAspIleAlaGlyValAlaLysIleAla    165170175    CGCAAGCACGGCGCGACCGTGGTGGTCGACAACACCTACTGCACGCCG636    ArgLysHisGlyAlaThrValValValAspAsnThrTyrCysThrPro    180185190    TACCTGCAACGGCCACTGGAGCTGGGCGCCGACCTGGTGGTGCATTCG684    TyrLeuGlnArgProLeuGluLeuGlyAlaAspLeuValValHisSer    195200205    GCCACCAAGTACCTGAGCGGCCATGGCGACATCACTGCTGGCATTGTG732    AlaThrLysTyrLeuSerGlyHisGlyAspIleThrAlaGlyIleVal    210215220    GTGGGCAGCCAGGCACTGGTGGACCGTATACGTCTGCAGGGCCTCAAG780    ValGlySerGlnAlaLeuValAspArgIleArgLeuGlnGlyLeuLys    225230235240    GACATGACCGGTGCGGTGCTCTCGCCCCATGACGCCGCACTGTTGATG828    AspMetThrGlyAlaValLeuSerProHisAspAlaAlaLeuLeuMet    245250255    CGCGGCATCAAGACCCTCAACCTGCGCATGGACCGCCACTGCGCCAAC876    ArgGlyIleLysThrLeuAsnLeuArgMetAspArgHisCysAlaAsn    260265270    GCTCAGGTGCTGGCCGAGTTCCTCGCCCGGCAGCCGCAGGTGGAGCTG924    AlaGlnValLeuAlaGluPheLeuAlaArgGlnProGlnValGluLeu    275280285    ATCCATTACCCGGGCCTGGCGAGCTTCCCGCAGTACACCCTGGCCCGC972    IleHisTyrProGlyLeuAlaSerPheProGlnTyrThrLeuAlaArg    290295300    CAGCAGATGAGCCAGCCGGGCGGCATGATCGCCTTCGAACTCAAGGGC1020    GlnGlnMetSerGlnProGlyGlyMetIleAlaPheGluLeuLysGly    305310315320    GGCATCGGTGCCGGGCGGCGGTTCATGAACGCCCTGCAACTGTTCAGC1068    GlyIleGlyAlaGlyArgArgPheMetAsnAlaLeuGlnLeuPheSer    325330335    CGCGCGGTGAGCCTGGGCGATGCCGAGTCGCTGGCGCAGCACCCGGCA1116    ArgAlaValSerLeuGlyAspAlaGluSerLeuAlaGlnHisProAla    340345350    AGCATGACTCATTCCAGCTATACCCCAGAGGAGCGTGCGCATTACGGC1164    SerMetThrHisSerSerTyrThrProGluGluArgAlaHisTyrGly    355360365    ATCTCCGAGGGGCTGGTGCGGTTGTCGGTGGGGCTGGAAGACATCGAC1212    IleSerGluGlyLeuValArgLeuSerValGlyLeuGluAspIleAsp    370375380    GACCTGCTGGCCGATGTGCAACAGGCACTCAAGGCGAGTGCC1254    AspLeuLeuAlaAspValGlnGlnAlaLeuLysAlaSerAla    385390395    TGAACCCGTCACGGATGAGGTCAATGCAATGGTGGCAATGATGAACCTTGTGCCTGGCGA1314    CGGCGT1320    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 398 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetHisGlySerAsnLysLeuProGlyPheAlaThrArgAlaIleHis    151015    HisGlyTyrAspProGlnAspHisGlyGlyAlaLeuValProProVal    202530    TyrGlnThrAlaThrPheThrPheProThrValGluTyrGlyAlaAla    354045    CysPheAlaGlyGluGlnAlaGlyHisPheTyrSerArgIleSerAsn    505560    ProThrLeuAsnLeuLeuGluAlaArgMetAlaSerLeuGluGlyGly    65707580    GluAlaGlyLeuAlaLeuAlaSerGlyMetGlyAlaIleThrSerThr    859095    LeuTrpThrLeuLeuArgProGlyAspGluValLeuLeuGlyAsnThr    100105110    LeuTyrGlyCysThrPheAlaPheLeuHisHisGlyIleGlyGluPhe    115120125    GlyValLysLeuArgHisValAspMetAlaAspLeuGlnAlaLeuGlu    130135140    AlaAlaMetThrProAlaThrArgValIleTyrPheGluSerProAla    145150155160    AsnProAsnMetHisMetAlaAspIleAlaGlyValAlaLysIleAla    165170175    ArgLysHisGlyAlaThrValValValAspAsnThrTyrCysThrPro    180185190    TyrLeuGlnArgProLeuGluLeuGlyAlaAspLeuValValHisSer    195200205    AlaThrLysTyrLeuSerGlyHisGlyAspIleThrAlaGlyIleVal    210215220    ValGlySerGlnAlaLeuValAspArgIleArgLeuGlnGlyLeuLys    225230235240    AspMetThrGlyAlaValLeuSerProHisAspAlaAlaLeuLeuMet    245250255    ArgGlyIleLysThrLeuAsnLeuArgMetAspArgHisCysAlaAsn    260265270    AlaGlnValLeuAlaGluPheLeuAlaArgGlnProGlnValGluLeu    275280285    IleHisTyrProGlyLeuAlaSerPheProGlnTyrThrLeuAlaArg    290295300    GlnGlnMetSerGlnProGlyGlyMetIleAlaPheGluLeuLysGly    305310315320    GlyIleGlyAlaGlyArgArgPheMetAsnAlaLeuGlnLeuPheSer    325330335    ArgAlaValSerLeuGlyAspAlaGluSerLeuAlaGlnHisProAla    340345350    SerMetThrHisSerSerTyrThrProGluGluArgAlaHisTyrGly    355360365    IleSerGluGlyLeuValArgLeuSerValGlyLeuGluAspIleAsp    370375380    AspLeuLeuAlaAspValGlnGlnAlaLeuLysAlaSerAla    385390395    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    IleHisHisGlyTyrAspPro    15    __________________________________________________________________________


7. A pharmaceutical composition comprising the enzyme as claimed in 5 or6 and a pharmaceutically acceptable carrier thereof.
 8. The recombinantprotein as claimed in claim 1 or 2 which can be purified from culturedhost cells transformed with a recombinant vector containing a nucleotidesequence from No. 61 or 64 to No. 1254 of that shown in SEQ ID NO:1, ora nucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence from No. 61 or 64 to No. 1254 of that shown in SEQID NO:1.
 9. An anti-tumor agent which comprises the enzyme as claimed inclaim 6.